Abstract The goal of the proposed research is to decipher a diversity of unexplored behavioral roles for rhodopsins in Drosophila melanogaster, and in the major disease vector, Aedes aegypti. The proposed studies build on our recent discovery that a fly rhodopsin (Rh7) acts as a light sensor in a small set of neurons in the brain, where it contributes to circadian photoentrainment. This represents the first function for a rhodopsin in the central brain, despite decades-old reports that rhodopsins are expressed in the brains of many animals, including humans. We propose to bring to bear a wide set of tools to reveal the rhodopsins and signaling proteins employed in the fly brain and eye for circadian photoentrainment and for sleep. We will then leverage our extensive background in flies and our recent expansion into mosquitoes to reveal the mosquito rhodopsins required for circadian photoentrainment and for detecting humans under different light conditions. This goal is important, since mosquitoes rely heavily on vision to identify humans. Yet, there are no molecular genetic studies focusing on vision or rhodopsins in any insect vector. The project?s success will be made possible by our development and application of an extensive suite of in vivo approaches including electrophysiology, behavioral assays, cell biology, molecular genetic approaches, and expertise in creating gene knockouts in Aedes. We propose to capitalize on a transformative technical innovation we have developed to accelerate molecular genetics in mosquitoes. Aim 1 will test the hypothesis that Rh7 in the fly brain functions in circadian photoentrainment through a signaling cascade distinct from the one used in the compound eye. The experiments will also test the idea that Rh7 confers greater light sensitivity to pacemaker neurons than is possible through Cryptochrome, the other light sensor in the brain. Aim 2 focuses on illuminating the roles of different rhodopsins in the Drosophila eye and brain on sleep. We will address the concept that two rhodopsins in a small subset of photoreceptor cells in the compound eye are required for normal nighttime sleep, while Rh7 in the brain affects daytime sleep. In Aim 3, we will decipher roles for rhodopsins in Aedes that are required for increased visual attraction to humans, following exposure to CO2. We will clarify which rhodopsins are most important for host recognition at different intensities of light. Finally, we will unravel roles for two rhodopsins expressed in the Aedes brain. In summary, the proposed project will deepen our understanding of visual and non-visual rhodopsins that function in circadian photoentrainment and sleep. These are important goals, since our understanding of the mechanisms underlying sleep is rudimentary, and many diseases are exacerbated by impairments in this evolutionarily conserved behavior. Finally, given the importance of mosquito vision for identifying humans, we propose that unraveling the rhodopsins that contribute to human recognition offers to lead to creative CRISPR/Cas9-based approaches to control insect vectors and reduce disease.